U.S. patent application number 09/027956 was filed with the patent office on 2001-12-06 for novel multi-oligosaccharide glycoconjugate bacterial meningitis vaccines.
Invention is credited to CHONG, PELE, KLEIN, MICHEL, LINDBERG, ALF.
Application Number | 20010048929 09/027956 |
Document ID | / |
Family ID | 21840755 |
Filed Date | 2001-12-06 |
United States Patent
Application |
20010048929 |
Kind Code |
A1 |
CHONG, PELE ; et
al. |
December 6, 2001 |
NOVEL MULTI-OLIGOSACCHARIDE GLYCOCONJUGATE BACTERIAL MENINGITIS
VACCINES
Abstract
Multivalent immunogenic molecules comprise a carrier molecule
containing at least one functional T-cell epitope and multiple
different carbohydrate fragments each linker to the carrier
molecule and each containing at least one functional B-cell
epitope. The carrier molecule inputs enhanced immunogenicity to the
multiple carbohydrate fragments. The carbohydrate fragments may be
capsular oligosaccharide fragments from Streptococcus pneumoniae,
which may be serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F or 23F, or
Neisseria meningitidis, which may be serotype A, B, C, W-135 or Y.
Such oligosaccharide fragments may be sized from 2 to 5 kDa.
Alternatively, the carbohydrate fragments may be fragments of
carbohydrate-based tumor antigens, such as Globo H, Le.sup.Y or
STn. The multivalent molecules may be produced by random
conjugation or site-directed conjugation of the carbohydrate
fragments to the carrier molecule. The multivalent molecules may be
employed in vaccines or in the generation of antibodies for
diagnostic application.
Inventors: |
CHONG, PELE; (RICHMOND HILL,
CA) ; LINDBERG, ALF; (LYON, FR) ; KLEIN,
MICHEL; (WILLOWDALE, CA) |
Correspondence
Address: |
SIM & MCBURNEY
6TH FLOOR 330 UNIVERSITY AVENUE
TORONTO
M5G1R7
CA
|
Family ID: |
21840755 |
Appl. No.: |
09/027956 |
Filed: |
February 23, 1998 |
Current U.S.
Class: |
424/234.1 ;
424/236.1; 424/244.1; 424/249.1 |
Current CPC
Class: |
A61K 39/095 20130101;
Y02A 50/30 20180101; A61K 39/385 20130101; Y02A 50/466 20180101;
A61K 2039/6037 20130101; A61K 2039/645 20130101; Y02A 50/484
20180101; Y10S 530/807 20130101; A61P 31/04 20180101; G01N 33/56944
20130101; Y10S 530/81 20130101; A61K 39/092 20130101; A61P 29/00
20180101; A61P 19/00 20180101; Y02A 50/59 20180101; A61P 37/00
20180101; A61K 47/646 20170801 |
Class at
Publication: |
424/234.1 ;
424/236.1; 424/249.1; 424/244.1 |
International
Class: |
A61K 039/095; A61K
039/09; A61K 039/02 |
Claims
What we claim is:
1. A multivalent immunogenic molecule, comprising: a carrier
molecule containing at least one functional T-cell epitope, and
multiple different carbohydrate fragments each linked to the
carrier molecule and each containing at least one functional B-cell
epitope, wherein said carrier molecule imparts enhanced
immunogenicity to said multiple carbohydrate fragments.
2. The molecule of claim 1 wherein said carbohydrate fragments are
bacterial capsular oligosaccharide fragments.
3. The molecule of claim 2 wherein said capsular oligosaccharide
fragments are capsular oligosaccharide fragments of Streptocococcus
pneumoniae.
4. The molecule of claim 3 wherein said capsular oligosaccharide
fragments are derived from at least two capsular polysaccharides of
S. pneumoniae serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F.
5. The molecule of claim 4 wherein said carrier molecule is a
T-cell epitope-containing protein or protein fragment of S.
pneumoniae.
6. The molecule of claim 2 wherein said capsular polysaccharide
fragments are capsular oligosaccharids fragments of Neisseria
meningitidis.
7. The molecule of claim 6 wherein said oligosaccharide fragments
are derived from at least two capsular polysaccharides of N.
meningitidis Group A, B, C, W-135 and Y.
8. The molecule of claim 7 wherein said carrier molecule is a
T-cell epitope-containing protein or protein fragment of N.
meningitidis.
9. The multivalent immunogenic molecule of claim 2 wherein said
oligosaccharide fragments are sized from about 2 to about 5
kDa.
10. The multivalent immunogenic molecule of claim 1 wherein said
carrier molecule is an oligopeptide containing at least one
functional T-cell epitope.
11. The multivalent immunogenic molecule of claim 1 wherein said
carrier molecule is a carrier protein.
12. The multivalent immunogenic molecule of claim 11 wherein said
carrier protein is tetanus toxoid.
13. The multivalent immunogenic molecule of claim 1 wherein said
carbohydrate fragments are fragments of carbohydrate-based tumor
antigens.
14. The multivalent immunogenic molecule of claim 13 wherein the
tumor antigen is Globo H, Le.sup.Y or STn.
15. The multivalent immunogenic molecule of claim 1 produced by
site-directed glycoconjugation.
16. A method of forming a multivalent immunogenic molecule,
comprising: treating at least two different carbohydrate molecules
to obtain carbohydrate fragments thereof, and conjugating each of
the carbohydrate fragments to a carrier molecule.
17. The method of claim 16 wherein said carbohydrate molecules are
capsular polysaccharides of a bacteria and oligosaccharide
fragments of said capsular polysaccharide are selected sized from 2
to 5 kDa.
18. The method of claim 17 wherein said conjugating step is
effected by random conjugation of said oligosaccharide fragments to
said carrier molecule.
19. The method of claim 17 wherein said conjugating step is
effected by site-directed glycoconjugation.
20. The method of claim 19 wherein said site-directed
glycoconjugation is effected by first forming a multiple antigen
peptide as the carrier molecule anchored to a polymeric anchor
wherein at least two carrier peptide segments have different
terminal protecting groups, selectively removing one of the
protecting groups, coupling a first one of the oligosaccharide
fragments to the unprotected carrier peptide segment, selectively
removing another of the protecting groups, coupling a second one of
the oligosaccharide fragments to the unprotected carrier peptide
segment, and cleaving the resulting molecule from the polymeric
anchor.
21. An immunogenic composition for meningitis, comprising: (1) a
multiple pneumococcal glycoconjugate according to claim 3, (2) a
multiple meningococcal glycoconjugate according to claim 6, and (3)
an immunogenic synthetic PRP-peptide conjugate.
22. The immunogenic composition of claim 21 further comprising at
least one additional antigen.
23. The immunogenic composition of claim 21 wherein said multiple
pneumococcal glycoconjugate according to claim 3 is derived from at
least two capsular polysaccharides of S. pneumoniae serotypes 1, 4,
5, 6B, 9V, 14, 18C, 19F and 23F and said multiple meningococcal
conjugate according to claim 6 is derived from at least two
capsular polysaccharides of Neisseria meningitidis serotypes A, B,
C, Y and W.
24. A method of generating an immune response, which comprises
administering to a host an immunoeffective amount of an immunogenic
as claimed in claim 21.
25. A method of determining the presence of antibodies specifically
reactive with a multivalent immunogenic molecule as claimed in
claim 1: (a) contacting the sample with said multivalent
immunogenic molecule to produce complexes comprising the molecule
and any said antibodies present in the sample specifically reactive
therewith; and (b) determining production of the complexes.
26. The method of claim 25 wherein said multivalent immunogenic
molecule is a multiple pneumococcal glycoconjugate as claimed in
claim 3.
27. The method of claim 26 wherein said multiple pneumococcal
glycoconjugate is derived from at least two capsular
polysaccharides of S. pneumoniae serotypes 1, 4, 5, 6B, 9V, 14,
18C, 19F and 23F.
28. The method of claim 25 wherein said multiple pneumococcal
glycoconjugate is derived from meningococcal glycoconjugate as
claimed in claim 6.
29. The method of claim 28 wherein said multiple meningococcal
glycoconjugate is derived from at least two capsular
polysaccharides of N. meningitidis serotypes A, B, C, Y and W.
30. A method of determining the presence of multivalent immunogenic
conjugate molecule of claim 1 in a sample, comprising the steps of:
(a) immunizing a subject with the immunogenic conjugate molecule to
produce antibodies specific for the carbohydrate fragments; (b)
isolating the carbohydrate fragment specific antibodies; (c)
contacting the sample with the isolated multivalent immunogenic
molecule present in the sample and said isolated carbohydrate
fragment specific antibodies; and (d) determining production of the
complexes.
31. The method of claim 30 wherein said multivalent immunogenic
molecule is a multiple pneumococcal glycoconjugate as claimed in
claim 3.
32. The method of claim 31 wherein said multiple pneumococcal
glycoconjugate is derived from at least two capsular
polysaccharides of S. pneumoniae serotypes 1, 4, 5, 6B, 9V, 14,
18C, 19F and 23F.
33. The method of claim 30 wherein said multiple pneumococcal
glycoconjugate is derived from meningococcal glycoconjugate as
claimed in claim 6.
34. The method of claim 33 wherein said multiple meningococcal
glycoconjugate is derived from at least two capsular
polysaccharides of N. meningitidis serotypes A, B, C, Y and W.
35. A diagnostic kit for determining the presence of a multivalent
immunogenic molecule of claim 1 comprising: (a) the multivalent
immunogenic molecule; (b) means for contacting the multivalent
molecule with the sample to produce complexes comprising the
multivalent molecule and any said antibodies present in the sample;
and (c) means for determining production of the complexes.
36. The kit of claim 35 wherein said immunogenic conjugate molecule
of claim 1 is present in the form of the immunogenic composition of
claim 21.
37. A diagnostic kit for determining the presence of a multivalent
immunogenic molecule in a sample, comprising: (a) antibodies
specific for carbohydrate fragments of the multivalent immunogenic
molecule; (b) means for contacting the antibodies with the sample
to produce a complex comprising multivalent immunogenic molecule
and the antibodies; and (c) means for determining the production of
the complex.
38. The kit of claim 37 wherein said antibodies are antibodies to
the components of the immunogenic composition of claim 21.
Description
FIELD OF INVENTION
[0001] The present invention is related to the field of vaccines
and is particularly related to the development of novel
glycoconjugation technologies which can be used to prepare
glycoconjugates in which multi-oligosaccharides are covalently
linked to the same carrier protein.
BACKGROUND OF THE INVENTION
[0002] Haemophilus influenzae type b (Hib), Neisseria meningitidis
and Streptococcus pneumoniae are major causes of bacterial
meningitis in children under five years of age. All these bacteria
are protected from phagocytosis by a polysaccharidic capsule.
Antibodies induced against the capsular polysaccharide (CPs) of the
organism are protective in most cases. Effective Hib conjugate
vaccines in which Hib CPs, PRP, is linked to different carrier
proteins, such as diphtheria toxoid (PRP-D), tetanus toxoid
(PRP-T), CRM 197 (HbOC) and the outer membrane proteins of N.
meningitidis (PRP-OMP), have been developed. Four Hib conjugate
vaccines are now commercially available. New glycoconjugate
vaccines against N. meningitidis and S. pneumoniae are highly
recommended by the American College of Physicians.
[0003] The development of multivalent pneumococcal vaccines for the
prevention of both systemic and noninvasive pneumococcal diseases
in infants, the elderly and immune-compromised individuals has
gained increasing importance over the last decade. For more
detailed reviews of pneumococcal disease, epidemiology, or the
polysaccharide vaccine, numerous review articles are available
(ref. 1, various references are referred to in parenthesis to more
fully describe the state of the art to which this invention
pertains. Full bibliographic information for each citation is found
at the end of the specification, immediately preceding the claims.
The disclosure of these references are hereby incorporated by
reference into the present disclosure). Streptococcus pneumoniae is
a capsulated, gram-positive bacterium that is present as normal
flora in the human upper respiratory tract. It is a frequent and
major cause of pneumonia, meningitis, bacteremia and noninvasive
bacterial otitis media. Disease incidence is highest in infants and
the elderly. In the United States alone, the overall incidence of
systemic pneumococcal infections is estimated to be 50/100,000 in
the geriatric population and 160/100,000 in children less than 2
years old (refs. 2, 3). Case fatalities can be as high as
40,000/year, especially in the geriatric population. Many serotypes
of S. pneumoniae are developing resistance to conventional
antibiotic treatments. The incidence of otitis media in children
approaches 90% by the age of 5 and the peak incidence occurs at 6
to 15 months of age. It was estimated that over 1.2 million cases
of otitis media occur annually. Recent studies on the epidemiology
of pneumococcal disease (ref. 4) have shown that five serotypes
(6B, 14, 19F, 23F and 18C) of the 85 known serotypes account for 70
to 80% of pneumococcal disease in infants and that in the United
States, types 9V and 4 are ranked sixth and seventh. In Europe and
developing countries, types 1 and 5 are more prevalent than types 4
and 9V. Thus, a pneumococcal conjugate vaccine for the United
States should contain at least seven serotypes (4, 6B, 9V, 14, 18C,
19F, and 23F) to achieve a 75 to 85% coverage. Conjugate vaccine
formulations for Europe and elsewhere should include serotypes 1,
5, 6B, 14, 18C, 19F and 23F. A broad-spectrum multivalent
pneumococcal conjugate vaccine should then contain CPs from nine
serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F.
[0004] N. meningitidis is a gram-negative bacterium that has been
serologically classified into groups A, B, C, 29e, W135, X, Y and
Z. Additional groups (H, I, and K) were isolated in China and group
L was isolated in Canada. The grouping system is based on the
capsular polysaccharides of the organism. In contrast to the
pneumococcal vaccine, the composition of the meningococcal
polysaccharide vaccine has been greatly simplified by the fact that
fewer polysaccharides are required. In fact groups A, B, and C are
responsible for approximately 90% of cases of meningococcal
meningitis. Prevention of group A and C meningococcal meningitis
can be achieved by vaccination with a bivalent polysaccharide
vaccine. This commercial vaccine has been used successfully in
adults during the last decade to prevent major meningitis epidemics
in many parts of the world. However, there is a need to improve
this vaccine because a significant proportion of cases of
meningococcal meningitis are due to serotypes other than A and C.
Group B N. meningitidis is of particular epidemiologic importance,
but groups Y and W135 are also significant. Although a tetravalent
vaccine comprising groups A, C, W135, and Y polysaccharides is the
current meningococcal meningitis vaccine, it is not very effective
in young infants, since maturation of the immune response to most
capsular polysaccharides in infants occurs around the age of 2
years.
[0005] The Group B meningococcal polysaccharide is poorly
immunogenic in man. Two major reasons have been proposed to account
for this phenomenon. One is that the .alpha.-(2.fwdarw.8)-linked
sialic acid homopolymer is rapidly depolymerized in human tissue by
neuraminidase. The other one is that Group B capsular
polysaccharide is a polymer of N-acetylneuraminic acid (.alpha.
2->8 NeuNAc), and that the .alpha. 2->8 NeuNAc moiety is
found as a monomer and dimer on several glycoproteins and
gangliosides in adults and as a polymer of at least eight repeating
units in rat fetal and newborn tissues. Thus, this structure is
recognized as a "self" antigen by the human immune system. As a
result, the production of antibody is suppressed or because of this
molecular mimicry, a vaccine based on native Group B CPs might
induce auto-antibodies directed against the .alpha. 2-8 NeuNAc
moiety, and thus cause autoimmune diseases.
[0006] Since the Group B meningococcal CPs is not immunogenic in
humans, approaches have been pursued to increase its
immunogenicity. One approach uses non covalent complexes of Group B
CPs and outer membrane protein (OMPs). Such complexes are formed by
hydrophobic interaction between the hydrophobic regions of the OMPs
and the diacyl glycerol group at the reducing end of the CPs. Human
volunteers were given two doses of the complex at 0 and 5 weeks.
Most individuals responded with an increase in antibodies to group
B CPs. However, the second dose resulted in little or no increase
in antibody titres which subsequently declined over a period of 14
weeks. The antibodies with group B polysaccharide specificity were
limited to the IgM class and directed against determinants present
only on high molecular weight polysaccharides.
[0007] To improve the immunogenicity of Group B CPs, Jennings (ref.
5) prepared a Group B meningococcal-tetanus toxoid conjugate
(GBMP-TT) by covalently linking the CPs to tetanus toxoid (TT)
through its terminal non-reducing sialic acid using periodate
oxidized CPs. This procedure, however, did not result in any
significant enhancement in CPs immunogenicity. The antibody
response elicited in animals was found to be primarily directed
against the linkage point between the CPs and the protein
(GBMP-TT). Further improvement of the immunogenicity of group B CPs
involved its chemical modification. Jennings (Ref. 6) reported that
the N-acetyl groups of group B CPs could be selectively removed by
the action of a strong base at elevated temperature. The acetyl
groups were then replaced with N-propionyl groups by propionic
anhydride treatment to produce N-propionylneuraminic acid (.alpha.
(2->8) NeuPro) polymers. The N-propionylated CPs was first
periodate oxidized with sodium periodate, and then coupled to TT in
the presence of sodium cyanoborohydride to yield the chemically
modified GBMP-TT conjugate. Mice immunized with this conjugate
formulated in Freund's complete adjuvant (FCA), generated high
levels of cross-reactive IgG antibody against native group B CPs.
Murine anti-sera were found to be bactericidal for all group B
strains. However, further studies revealed the existence of two
populations of antibodies with different specificity. One
population reacted with purified group B CPs wherteas the other one
did not. Antibodies that did not react with native group B CPs
appeared to be responsible for bactericidal activity. These
antibodies may recognize an epitope expressed by cell-associated
CPs that is not present on purified CP. Alternative conjugates
comprising the capsular polysaccharide of N. meningitidis group B
CPs conjugated to a carrier protein as immunogenic compositions,
including vaccines, and their use as and for the generation of
diagnostic reagents, had been described by Kandil et al. (U.S.
patent application No. 08/474,392 filed Jun. 7, 1995, assigned to
the Assignee hereof and the disclosure of which is incorporated
herein by reference, EP 0747063). In particular, the capsular
polysaccharides of N. meningitidis contain multiple sialic
derivatives that can be modified and used to attach carrier
molecules.
[0008] The dramatic reduction in Haemophilus influenzae type b
disease observed in countries that have licensed and used Hib
CPs-protein conjugate vaccines, has demonstrated that CPs-protein
conjugates can prevent systemic bacterial diseases. It is
reasonable to expect that meningococcal and pneumococcal
CPs-protein conjugates will also be efficacious. The possibility of
preventing noninvasive diseases, such as otitis media, by systemic
immunization with conjugate vaccines needs to be explored. Whether
high titers of serotype-specific antibodies are sufficient to
prevent either nasopharyngeal colonization and/or otitis media
remains an open question. The development of an otitis media
vaccine requires a multiple pneumococcal CPs-protein conjugates to
elicit high anti-CPs antibody titers early in life.
[0009] The development of both multivalent pneumococcal and
meningococcal CPs-protein conjugate vaccines to prevent systemic
and noninvasive diseases presents many challenges to carbohydrate
chemists, immunologists, clinicians and vaccine manufacturers. The
amount of carbohydrate, the choice of carrier, the method of
vaccine delivery, and the use of immuno stimulants or adjuvants are
known to influence on the host immune responses. Immunogenic
glycoconjugates can be formed between multifunctionalized CPs and
proteins if the conditions are controlled very carefully. Most of
the conjugates are today synthesized by coupling either CPs or
oligosaccharides activated through the reducing end to a protein or
peptide with or without a linker group.
[0010] A general glycoconjugation method involves random activation
of the capsular polysaccharide or fragments of the polysaccharide
by periodate treatment. The reaction leads to a random oxidative
cleavage of vicinal hydroxyl groups of the carbohydrates with the
formation of reactive aldehyde groups. Coupling to a protein
carrier is by direct amination to the lysyl groups. A spacer group
such as aminocaproic acid, can be reacted with the aldehydes by
reductive amination and then coupled to the protein lysyl groups by
water soluble carbodiimide condensation (ref. 7). The
oligosaccharide-peptide conjugate reported by Paradiso (ref. 8) was
prepared similarly except that a peptide presenting a T-cell
epitope of CRM.sub.197 was used instead of the native protein.
Other conjugation approaches that have been disclosed by Gordon in
U.S. Pat. No. 4,496,538 and by Schneerson et al. (ref. 9), involve
directly derivatizing the CPs with adipic acid dihydrazide (ADH)
following CNBr activation, and then conjugating the derivatized CPs
directly to a carrier protein (D or T) by carbodiimide
condensation. Marburg and Tolman (EP#534764A1) demonstrated that
protein-dimeric CPs conjugate immunogens could be produced by
coupling the first CPs to a protein carrier and then linking the
second CPs to the first CPs via a bifunctional cross-linker.
[0011] Methods for inducing immunity against disease are constantly
improving. Research has focused on the structure-function
relationship of carbohydrate protein conjugates with the hope of
discovering the mechanisms of B- and T-cell interactions with
conjugates that could lead to vaccines with improved immunogenicity
and to the development of adjuvants and delivery systems. Chong et
al. (U.S. Pat. No. 5,679,352 assigned to the assignee hereof and
the disclosure of which is incorporated herein by reference) showed
that several factors affect the immunogenicity of carbohydrates.
The minimum requirements for the synthesis of an immunogenic
glycoprotein conjugate are that the B-cell epitope(s) of the CPs
and the T-cell epitope(s) of the carrier should be functional after
covalent linkage. The magnitude of the anti-CPs antibody response
markedly depends on the spatial orientation of CPs relative to the
T-cell epitopes. Anti-CPs antibody responses are enhanced when
multiple antigenic peptides (MAPs) are used as carriers.
[0012] A single-dose polyvalent vaccine is listed as the first
priority in the WHO vaccine development programme. A single-dose
polyvalent CPs-protein conjugate vaccine (15 different CPs-protein
conjugates: 1 Hib conjugate, five N. meningitidis conjugates and
nine S. pneumoniae conjugates) against bacterial meningitis,
presents a potential risk of hyperimmunization against classical
carrier proteins, such as diptheria and tetanus toxoids. It is
documented that non-epitope-specific suppression of the antibody
response to Hib conjugate vaccines by pre-immunization with carrier
proteins (ref. 11). Thus, appropriate approaches are required to
solve this vaccine formulation problem. Some of the problems can be
circumvented by incorporating conserved, cross-protective,
non-capsular antigens from Hib, N. meningitides and S. pneumoniae.
Although several outer membrane proteins have been proposed as
vaccine candidates, none of them has been tested in clinical
trials. A multiple CPs-carrier conjugate delivery system thus
represents a novel generic approach and will be important in
glycoconjugate vaccine development. Therefore, the present
invention is directed towards novel glyconjugation technologies
which can be used to prepare vaccines containing multiple
oligosaccharides from different bacteria covalently linked to the
same carrier protein or polypeptides.
SUMMARY OF THE INVENTION
[0013] In accordance with one aspect of the present invention,
there is provided a multivalent immunogenic molecule, comprising a
carrier molecule containing at least one functional T-cell epitope,
and multiple different carbohydrate fragments each linked to the
carrier molecule and each containing at least one functional B-cell
epitope, wherein said carrier molecule imparts enhanced
immunogenicity to said multiple carbohydrate fragments.
[0014] In one embodiment of the invention, the carbohydrate
fragments are bacterial capsular oligosaccharide fragments. Such
capsular polysaccharide fragments may be oligosaccharide fragments
of Streptococcus pneumoniae, including fragments derived from at
least two capsular polysaccharide of S. pneumoniae serotypes 1, 4,
5, 6B, 9V, 14, 18C, 19F and 23F. The carrier molecule may be a
T-cell epitope-containing protein or protein fragment of S.
pneumoniae.
[0015] The capsular polysaccharide fragments may be oligosaccharide
fragment of Neisseria meningitidis, including fragments derived
from at least two capsular polysaccharide of N. meningitidis Group
A, B, C, W-135 and Y. The carrier molecule may be a T-cell
epitope-containing protein or protein fragment of N.
meningitidis.
[0016] The capsular polysaccharide employed in this aspect of the
invention may be employed as oligosaccharide fragments sized from
about 1 to about 5 kDa. Such fragments may be provided by acid
hydrolysis of the capsular polysaccharide. The oligosaccharide
fragments may be chemically modified for coupling to the carrier
molecule.
[0017] The carrier molecule may be an oligopeptide containing at
least one functional T-cell epitope or a carrier protein, such as
tetanus toxoid.
[0018] Tin another embodiment of the invention, the carbohydrate
fragments are fragments of carbohydrate-based tumor antigens. Such
carbohydrate-based tumor antigen may be Globo H, Le.sup.Y or STn.
In accordance with another aspect of the invention, there is
provided a method of forming a multivalent immunogenic molecule,
comprising treating at least two different carbohydrate molecules
to obtain carbohydrate fragments thereof, and conjugating each of
the carbohydrate fragments to a carrier molecule.
[0019] In one embodiment, the carbohydrate molecule is a capsular
polysaccharide of a bacteria and oligosaccharide fragments of the
capsular polysaccharide are selected sized from 2 to 5 kDa. Such
oligosaccharide fragments generally are derived from at least two
different serotypes of the same bacteria, including S. pneumoniae
and N. meningitidis.
[0020] In this embodiment of the present invention, such
multivalent immunogenic molecules may be provided by
glycoconjugation wherein three or more chemically-activated
capsular polysaccharides or their derivations simultaneously to a
single carrier molecule, providing a random conjugation. This
procedure is illustrated in FIG. 1.
[0021] In this embodiment of the invention, rational design of
lysine-branched peptide systems may be employed for site-directed
glypoconjugation. Using different side-chain protecting groups for
lysine and cysteine residues during peptide synthesis, activated
oligosaccharides may be selectively and sequentially linked to the
same carrier through such residues. This procedure is illustrated
in FIG. 2.
[0022] The method of site-directed conjugation may comprise first
forming a multiple antigen peptide as the carrier molecule and
anchored to a polymeric anchor wherein at least two carrier peptide
segments have different terminal protecting groups. One of the
protecting groups then is selectively removed and a first one of
the oligosaccharide fragments is coupled to the unprotected carrier
peptide segment. Another of the protecting groups is selectively
removed and a second one of the oligosaccharide fragments to the
unprotected carrier peptide segment. This procedure may be repeated
for as many carrier peptides as is provided. The resulting molecule
is claimed from the polymeric anchor.
[0023] In accordance with a further aspect of the invention, there
is provided an immunogenic composition for meningitis, comprising
(1) a multiple pneumococcal glycoconjugate according to claim 3,
(2) a multiple meningococcal glycoconjugate according to claim 6,
and (3) an immunogenic synthetic PRP-peptide conjugate.
[0024] The multiple pneumococcal glycoconjugate may be derived from
at least two capsular polysaccharides of S. pneumoniae serotypes 1,
4, 5, 6B, 9V, 14, 18C, 19F and 23F. The multiple meningococcal
glycoconjugate may be derived from at least two capsular
polysaccharides of N. Meningitidis Groups a, B, C, W-135 and Y.
[0025] Such universal meningitis immunogenic composition may be
combined with other antigens, such as DTP-polio, to provide a
polyvalent vaccine.
[0026] The present invention further includes a method of
generating an immune response in a host by administering to the
host an immunoeffective amount of an immunogenic composition of the
present invention.
[0027] The present invention further includes diagnostic procedures
and kits using the multivalent immunogenic molecule herein.
Accordingly, in an additional aspect of the invention, there is
provided a method of determining the presence of antibodies
specifically reactive with a multivalent immunogenic molecule as
provided herein:
[0028] (a) contacting the sample with said multivalent immunogenic
molecule to produce complexes comprising the molecule and any said
antibodies present in the sample specifically reactive therewith;
and
[0029] (b) determining production of the complexes.
[0030] In a further aspect of the invention, there is provided A
diagnostic kit for determining the presence of a multivalent
immunogenic molecule as provided herein, comprising:
[0031] (a) the multivalent immunogenic molecule;
[0032] (b) means for contacting the multivalent molecule with the
sample to produce complexes comprising the multivalent molecule and
any said antibodies present in the sample; and
[0033] (c) means for determining production of the complexes.
[0034] The present invention, therefore, permits pneumococcal
glycopeptide conjugates to be used in a diagnostic immunoassay
procedure or kit to detect the presence of anti-pneumococcal
protein and CPs antibodies, for example, anti-CPs 1, 4, 5, 6B, 9V,
14, 18C, 19F and 23F and anti-pneumococcal surface protein A
antibodies, or anti-meningococcal protein and CPs antibodies, for
example, anti-CPs A, B, C, Y and W-135 and anti-meningococcal OMP
class 1 antibodies.
[0035] In an additional aspect of the invention, there is provided
a method of determining the presence of multivalent immunogenic
conjugate molecule in a sample, comprising the steps of:
[0036] (a) immunizing a subject with the immunogenic conjugate
molecule to produce antibodies specific for the carbohydrate
fragments;
[0037] (b) isolating the carbohydrate fragment specific
antibodies;
[0038] (c) contacting the sample with the isolated multivalent
immunogenic molecule present in the sample and said isolated
carbohydrate fragment specific antibodies; and
[0039] (d) determining production of the complexes.
[0040] A further aspect of the invention provides a diagnostic kit
for determining the presence of a multivalent immunogenic molecule
in a sample, comprising:
[0041] (a) antibodies specific for carbohydrate fragments of the
multivalent immunogenic molecule;
[0042] (b) means for contacting the antibodies with the sample to
produce a complex comprising multivalent immunogenic molecule and
the antibodies; and
[0043] (c) means for determining the production of the complex.
[0044] The present invention also extends to the use of a mixture
of anti-PRP, anti-pneumococcal CPs and anti-meningococcal CPs
antibodies as a component in a diagnostic immunoassay kit to detect
the presence of Hib, S. pneumoniae and N. meningitidis in
biological specimens.
BRIEF DESCRIPTION OF THE FIGURES
[0045] The invention will be further understood from the following
descriptions and specific Examples with reference to the Figures in
which:
[0046] FIG. 1 shows a schematic diagram of several pneumococcal CPs
randomly conjugated to a carrier protein, such as TT, and the
procedure employed.
[0047] FIG. 2 shows a schematic diagram of the sequential
cross-linking of chemically activated pneumococcal oligosaccharides
to a lysine-branched peptide containing several functional T-cell
epitopes from pneumococcal proteins.
[0048] FIG. 3 shows the elution profile obtained during
purification of acid-hydrolysed oligosaccharides of S. pneumonaie
14 using gel permeation chromatography on a Sephadex.RTM.-G100
column.
[0049] FIG. 4 shows the elution profile obtained during
purification of the acid-hydrolysed oligosaccharides of N.
meningitidis group B using a Sephadex.RTM.-G100 gel permeation
chromatography.
[0050] FIG. 5 shows an elution profile obtained during purification
of multivalent S. pneumoniae oligosaccahrides-TT conjugates.
[0051] FIG. 6 shows rabbit antibody responses to multivalent S.
pneumoniae oligosaccahrides-TT conjugates formulated in FCA.
[0052] FIG. 7 shows rabbit antibody responses to multivalent S.
pneumoniae oligosaccahrides-TT conjugates formulated in alum.
[0053] FIG. 8 shows mouse antibody responses to multivalent S.
pneumoniae oligosaccahrides-TT conjugates formulated in FCA.
[0054] FIG. 9 shows rabbit antibody responses to multivalent N.
meningitidis oligosaccahrides-TT conjugates formulated in FCA.
[0055] FIG. 10 shows rabbit antibody responses to multivalent S.
pneumoniae glycopeptide conjugates formulated in FCA.
[0056] FIG. 11 shows rabbit antibody responses to multivalent S.
pneumoniae oligosaccahrides-MAP conjugates formulated in FCA.
GENERAL DESCRIPTION OF INVENTION
[0057] As discussed above, the present invention is related to
novel glycoconjugation technologies that can be used to covalently
link either multiple oligosaccharides from bacteria such as H.
influenzae, N. meningitidis, S. pneumoniae, E. coli, and Group B
Streptococcus, or carbohydrate-based tumor antigens, to the same
carrier protein or polypeptide(s) and to the multivalent molecules
produced thereby.
[0058] The development of strong and long-lasting humoral immunity
requires the recognition of foreign antigens by at least two
separate subsets of lymphocytes. B-lymphocytes (B-cells,
lymphocytes derived from bone marrows), are the precursors of
antibody-forming cells, and T-lymphocytes (T-cells, lymphocytes
derived from thymus) modulate the function of B-cells. Most CPs are
T-cell independent antigens and are capable of directly stimulating
B-cells to produce antibodies. In general, CPs induce B-cells to
terminally differentiate into antibody-secreting cells (plasma
cells), but antibody responses are short-lived and limited by the
number of responsive B-cells. Proteins and peptides are T-cell
dependent antigens, and contain epitope(s) that can form
peptide:MHC class II complexes on a B-cell and trigger armed helper
T-cells to synthesize both cell-bound and secreted cytokines
(effector molecules) that synergize in B-cell activation and clonal
expansion. CPs can be converted into T-dependent antigens by
coupling to a carrier protein or T-cell epitope(s) (ref. 9; U.S.
Pat. No. 4,496,538). By repeated immunization with CPs-protein
conjugates, the B-cell population in the vaccinees enters not only
antibody production, but also proliferation and maturation. As a
results, there are more B-cell making anti-CPs antibodies and
higher antibody titers as booster responses.
[0059] Rationale for using oligosaccharides as antigens
[0060] The minimum requirements for producing immunogenic
glycoprotein conjugates are that the B-cell epitope(s) of the CPs
and the T-cell epitope(s) of the carrier are functional after
covalent attachment. To randomly conjugate two or more CPs to the
same carrier protein or T-cell epitope(s), the size of the
carbohydrate is reduced to about 2 kDa to about 5 kDa to prevent
steric hindrance effects. At least two different approaches can
used to covalently link multiple oligosaccharides to a carrier
protein. The first approach is to activate or derivatize the
oligosaccharides using the same chemistry, so that their
conjugation to the carrier can be achieved simultaneously (FIG. 1).
The second approach uses lysine-branched peptide systems for
site-directed glycoconjugation. Using different side-chain
protecting groups for lysine and cysteine residues during peptide
synthesis, the activated oligosaccharides can be selectively and
sequentially coupled to the same carrier protein via these residues
(FIG. 2).
[0061] Preparation of oligosaccharides
[0062] As described in detail in the Examples below, acid
hydrolysis of various serotypes of Streptococcus pneumoniae
capsular polysaccharides (>50 kDa) may be carried out to form
oligosaccharides with a molecular weights ranging from about 2 to
about 5 kDa. This process may comprise three steps: (1) acid
hydrolysis of CPs in sealed vial under argon, (2) lyophilization
and (3) purification of oligosaccharides by gel-filtration
chromatography. The protocol for acid hydrolysis of CPs from S.
pneumoniae serotypes 1, 4, 5, 9V and 14 has been optimized.
Typically, CPs (2 mg/mL) are incubated in 0.5M trifluoroacetic acid
(TFA) at about 50.degree. to about 90.degree. C. for about 5 to
about 10 hours. Since CPs from serotypes 6B and 19F contain labile
phosphodiester bonds, their hydrolysis is performed under mild acid
conditions (about 10 to about 50 mM acetic acid) at about
50.degree. to about 100.degree. C. for about 30 to about 48 hours.
The CPs of serotype 23F can be partially hydrolyzed by either
incubating in about 0.1 to about 0.5M trifloroacetic acid (TFA) at
about 70.degree. C. for about 2 to about 4 hours or in about 1 to
about 50 mM acetic acid at about 80.degree. to about 110.degree. C.
for about 40 to about 60 hours. At the end of each hydrolysis, the
reaction solutions are diluted 5-fold with water, then lyophilized.
The purification of the crude oligosaccharides can be accomplished
using Sephadex.RTM. G-100 gel filtration chromatography (about
2.times.about 210 cm column). Typical chromatographic results are
illustrated in FIG. 3. The fractions are assayed for the presence
of carbohydrate using the resorcinol/sulfuric acid assay (Ref. 1).
The elution profile is plotted, and the chromatographically
purified oligosaccharides with a mean mass of about 2 to about 5
kDa are pooled. Molecular weight markers used to calibrate the
column are: dextran standards (39,100 and 8,800 Da), synthetic PRP
hexamer (2,340 Da), sucrose (342 Da) and glucose (180 Da). Sized
oligosaccharides of about 2 to about 5 kDa contain about 4 to about
8 repeating units in general and are expected to contain at least
one B-cell epitope. The yields are about 70 to about 90%. These
chromatographically purifed oligosaccharides are then used to
prepare glycoconjugates comprised of multiple-oligosaccharides
covalently linked to a carrier protein or a multiple antigen
peptide system (MAP) containing T-cell epitopes from S.
pneumococcal proteins.
[0063] As described in detail in the Examples below, acid
hydrolysis of various serotypes of N. meningitidis capsular
polysaccharides (>10 kDa) may be carried out to form
oligosaccharides with a mean molecular weight of about 2 to about 5
kDa. In common with the acid hydrolysis of pneumococcal CPs, the
process comprises acid hydrolysis, lyophilization and purification
using gel-filtration chromatography. The conditions for acid
hydrolysis of CPs from N. meningitidis groups C, W-135 and Y have
been optimized. Typically, CPs (10 mg/mL) are mixed with about 20
to about 80 mM sodium acetate, pH about 4.5 to about 5.5, in sealed
vials under argon at about 65.degree. to about 100.degree. C. for
about 8 to about 12 hours. Since group B CPs can undergo
intramolecular esterification under acidic conditions, The
conditions used for CPs group C hydrolysis are employed, but the
incubation time is limited to about 1 hr and the pH of the reaction
is immediately adjusted to pH 7 with about 0.1M NaOH to reverse the
esterification process. Group A CPs contain labile phosphodiester
bonds, thus they are hydrolyzed under mild acidic condition (such
as about 10 to about 20 mM acetic acid) and incubated at about
50.degree. to about 100.degree. C. for about 30 to about 48 hours.
At the end of each hydrolysis, the reaction solutions are diluted
5-fold with water and then lyophilized. The crude oligosaccharides
are fractioned by Sephadex.RTM. G-100 gel filtration chromatography
(about 2.times.about 210 cm column, see above). Typical
chromatographic results are illustrated in FIG. 4. The fractions
are assayed for the presence of siallc acid using the
resorcinol/sulfuric acid assay (ref. 12). The elution profile is
plotted, and chromatographically purified oligosaccharides of about
2 to about 5 kDa are pooled. Sized oligosaccharides typically
contain about 6 to about 15 repeating units and are expected to
contain at least one B-cell epitope. The yields are about 40 to
about 80%. These chromatographically-purifed oligosaccharides are
used to prepare glycoconjugates comprised of
multiple-oligosaccharide- s covalently linked to a carrier protein
or a multiple antigen peptide system (MAP) containing T-cell
epitopes from meningococcal proteins.
[0064] Similar procedure may be used for capsular polysaccharides
of other bacteria.
[0065] Carrier selection.
[0066] Although several pneumococcal and meningococcal membrane
proteins, such as pneumolysin (ref. 13), pneumococcal surface
protein A (PspA) (ref. 14), S. pneumoniae 37 kDa protein (SP37)
(ref. 15), meningococcal transferrin-binding protein 2 (Tbp2) (ref.
16), meningococcal pilin (ref. 17), and class 1 proteins (ref. 18),
have been identified as potential protective antigens, none of them
so far has been tested in clinical trials. These proteins contain
potential T-cell epitopes which have been identified using
conventional algorithms. Therefore, a panel of potential peptide
carriers may be selected for conjugation with the meningococcal and
pneumococcal oligosaccharides to form the multivalent immunogenic
molecules herein.
[0067] In the present invention, peptides (Table I; SEQ ID NOS: 1
to 8) to be coupled to oligosaccharides were chosen on the basis of
either their potential T-helper cell stimulatory properties or
their potential protective ability or the conservation of sequences
that would be important to recall T-cell memory. NMTBP2 (SEQ ID NO:
1) is a peptide fragment of N. meningitidis Tbp2 protein and had
previously been identified to contain both functional T-cell
epitope(s) and a strain-specific protective B-cell epitope
recognized by a Tbp2-specific MAb (U.S. patent application No.
08/337,483, assigned to the assignee hereof and the disclosure of
which is incorporated herein be reference; W095/13370). Peptides
NMC-1 and -2 (SEQ ID NOS: 2 and 3) were identified to contain the
immunodominant human T-cell epitopes of N. meningitidis class 1
protein (ref. 19). NMPi-1 (SEQ ID NO: 4) was derived from N.
meningitidis pilin protein and shown to contain sequences involved
in adhesion (ref. 17). Peptides PN(123-140; SEQ ID NO: 5) and
PN(263-281; SEQ ID NO: 6) derived from S. pneumoniae pneumolysin,
both contain with funtional T-cell epitopes (ref. 20). SP37 (SEQ ID
NO: 7) is the N-terminal fragment of from S. pneumoniae 37 kDa
protein and shown to be highly immunogenic in rabbit immunogenicity
studies. PSP-AA (SEQ ID NO: 8) is the N-terminal fragment of from
S. pneumoniae PSPA protein and shown to be capable of eliciting
protective immune responses in mice against live pneumococcal
bacterial challenge (ref. 14).
[0068] Immunogenicity of multi-oligosaccharide-carrier conjugates
in animal models.
[0069] A. Random conjugation approach (FIG. 1)
[0070] In the present invention, acids have been used to hydrolyze
bacterial capsular polysaccharides to low-molecular-weight
oligosaccharide fragments. The oligosaccharides can be purified and
reacted with either ammonia or diaminoethane to generate a free
terminal amino group at their reducing ends. The amino groups are
reacted with an excess of the disuccinimidyl ester of adipic acid
to introduce an active succinimidyl ester group. The activated
oligosaccharides are then reacted with the free amino groups of
carrier proteins or peptides to form covalent amide bonds. The
glycoconjugates comprise at least two oligosaccharides coupled per
protein/peptide molecule.
[0071] To avoid anti-linker antibody responses, oligosaccharides
can be directly coupled to the carriers using the reductive
amination procedure described by Jennings and Lugowsky (ref. 6).
Advantages of this latter procedure are that linker molecules are
unnecessary, thus eliminating the formation of potential neoantigen
groups, and that a very stable secondary amine or in some cases a
tertiary amine linkage, is formed between the saccharide and the
protein. In addition, treatment of most meningococcal and
pneumococcal capsular polysaccharides with periodate does not cause
a reduction in molecular weight of the polysaccharide or fragment
since oxidation takes place either on branch side chains or on
cyclic sugar residues of the main chain. In either case, the main
chain is not cleaved and the molecular size remains intact.
[0072] To evaluate the potential use of the multivalent molecules
of the present invention, oligosaccharides from S. pneumoniae
serotypes 6B, 14, 19F and 23F were randomly and covalently linked
to TT, as shown in FIG. 1. The resulting multiantigenic
glycoconjugate (MAG) was purified by the gel-filtration
chromatography (FIG. 5). Protein and carbohydrate analyses revealed
that the carbohydrate to protein molar ratio was 7.1:1. Four
individual conjugates (6-TT, 14-TT, 19F-TT and 23F-TT) were
prepared from fragments of the four respective serotypes with the
same method for comparative studies. The multiple antigenic
glycoconjugate (MAG) was formulated either with Freund's complete
adjuvant (FCA) or alum. Results from rabbit and mouse (BALB/c)
immunogenicity studies indicated that:
[0073] a. Strong antibody responses to all four serotype CPs were
observed in rabbits when FCA was used as adjuvant (FIG. 6). Titers
were comparable to those obtained with individual conjugates.
[0074] b. When alum was used as adjuvant in rabbits, only anti-14,
-19F and -23F antibody responses were observed and no anti- 6B was
found (FIG. 7).
[0075] c. Only anti-14 and 19F antibodies were elicited in BALB/c
mice (FIG. 8).
[0076] The biological activity of anti-pneumococcal antibodies can
be assayed by two different methods: in vitro opsonophagocytic
assays and in vivo animal protection studies using either active or
passive immunization. Previous studies (refs. 21 and 22) have shown
that anti-S. pneumococcal CPs antibodies were biologically active
and protective. There was a direct correlation between the total Ig
antibody ELISA titers and opsonization titers. Therefore, the
pneumococcal MAG candidate vaccine which can elicit anti-S.
pneumoniae CPs antibody responses in animal models, will be useful
for human immunization.
[0077] An N. meningitidis glycoconjugate containing group C, W and
Y oligosaccharides was prepared as described above following the
procedure shown schematically in FIG. 1. The multiple antigenic
glycoconjugate was purified by gel-filtration chromatography. The
molar ratio of carbohydrate to protein was found to be 6.6:1.
Rabbit immunogenicity studies revealed that meningococcal MAG could
elicit antibody responses against all three polysaccharides (groups
C, W and Y) in carbohydrate-specific ELISAs (FIG. 9), and that the
antisera had no reactivity against S. pneumoniae 6B PC used as
negative control. The reactivities of antibodies against groups W
and Y were very similar (geometric mean titer (GMT) about 3000).
Group C was less immunogenic in this multivalent glycoconjugate,
with a GMT about 500.
[0078] B. Multiple antigenic peptide (MAP) approach (FIG. 2).
[0079] In this invention, we provide methods to design and
synthesize novel lysine-branching peptides containing different
T-helper cell epitopes (multiple antigenic peptide, MAP) to which
several different oligosaccharides can be selectively and
sequentially coupled. To test this concept, resin-bound MAP was
synthesized and characterized as shown below. 1
[0080] A Fmoc-Lys (t-Boc) -TGA resin (500 mg, purchased from
BACHEM) with a substitution level of 180 .mu.mol/g was used to
prepare MAP. A standard Fmoc chemistry coupling protocol was used
(4-fold excess of Fmoc-protected amino acids,
O-benzotriazoyl-N,N,N',N'-tetramethyluronium hexafluorophosphate
(HBTU) and N-hydroxybenzotriazoie (HOBT) Idusopropylethylamine
(DIEA) for 1 hr (Example 6)). In order to facilitate the
conjugation of oligosaccharides, the substitution level of MAP was
reduced to about 50 .mu.mol/g when the first Fmoc-Gly residue was
coupled. When the synthesis was completed, a small portion of
MAP-resin was cleaved with 95% trifluroacetic acid (TFA) in the
presence of ethane dithiol (EDT) and thioanisol. Amino acid
analysis revealed that the cleaved MAP had the correct amino acid
compositions.
[0081] MAP (150 mg) was treated with dithioanisol (DTT) in dimethyl
formamide (DMF) to remove the trityl group from cysteine residues
to conjugate oligosaccharides derivatized with SH-directed
functional groups, such as m-maleimidobenzoyl-N-hydroxysuccinimide
(MBS). After reduction, the resulting MAP resins were then assayed
for amino groups and sulfydryl groups. From Ellam's assay the SH
substitution level was found to be 64 .mu.mol per g of MAP resin,
and the degree of substitution of amino groups was found to be 71
.mu.mol/g from the ninhydrin assay. These results indicated that
trityl and Fmoc protecting groups could be quantitatively
removed.
[0082] (PRP).sub.6-MBS which was prepared from a synthetic hexamer
of 3-.beta.-D-ribose-(1-1)-D-ribitol-5-phosphate derivatized with
MBS (ref. 23), was dissolved in DMF/PBS solution and then coupled
to the fully deprotected and reduced MAP under degassed conditions.
After coupling, the MAP-(PRP).sub.6 was subjected to the Ellman's
test for sulfhydryl group determination. The level of SH
substitution was reduced to 6.85 .mu.mol per g of resin. The
coupling of PRP was independently confirmed by the ribose assay and
was found to be 18 .mu.g of (PRP).sub.6/mg resin.
[0083] The resulting MAP-PRP.sub.6 was mixed with periodate
oxidized S. pneumoniae serotype 19F (1 eq.) in methanol/phosphate
buffer (pH 7.8) in the presence of NaCNBH.sub.3 at 38.degree. C.
for 6 days. After conjugation, the amino group substitution
determined by the ninhydrin assay was found to be 16 .mu.mol per g
of resin. Total sugar content was again found to be 16.1 mg/g
resin. A small portion of 19F-(PRP).sub.6 -MAP glycoconjugate-resin
was cleaved with 95% TFA in the presence of ethane dithiol (EDT)
and thioanisol. After the work-up, the MAP-glycoconjugate was found
to have the correct amino acid composition and carbohydrate
content. These results strongly suggest that different
oligosaccharides can be selectively and sequentially conjugated to
MAP resin.
[0084] Before synthesizing a MAP resin containing different T-cell
epitopes, periodate-oxidized pneumococcal oligosaccharides from
serotypes 6B, 14 and 23F were tested for coupling efficiently to
resin-bound linear peptides corresponding either to PN(123-140)(SEQ
ID NO: 5) or PN(263-281) (SEQ ID NO: 6), which are T-cell epitopes
derived from S. pneumoniae membrane protein pneumolysin. Linear
glycopeptides 6B-PN(123-140), 14-PN(263-281) and 23F-PN(123-140)
were prepared using reductive amination. The coupling efficiency of
oligosaccharide to resin-bound peptide was found to be 10 to 30% as
judged by the free amino group determination using the ninhydrin
assay. The glycopeptides were cleaved from the resin using 95% TFA,
then semi-purified by RP-HPLC. Rabbit immunogenicity studies were
performed with an "equimolar" combination of these linear
glycopeptides formulated in FCA/IFA. The results indicated that the
glycopeptide conjugates were immunogenic and elicited anti-6B,
anti-14 and anti-23F polysaccharide antibody responses (FIG. 10).
In addition, rabbit antisera reacted with the peptides as judged by
peptide-specific ELISAs (Table 2).
[0085] A MAP resin containing three T-cell epitopes derived from
different S. pneumococcal membrane proteins was synthesized using a
Fmoc-Gly-Lys-TGA resin with a substitution level of 50 .mu.mol/g,
as shown in the FIG. 2. The whole synthesis was carried out
manually using the optimized Fmoc chemistry coupling protocol
described above. When the synthesis was completed, a small portion
of MAP-resin was cleaved with 95% TFA in the presence of EDT and
thioanisol, the cleaved MAP was found to have the correct amino
acid compositions by amino acid analysis. The MAP resin was reduced
by DTT to remove the t-butylthio protecting groups from the
cysteine residues. After excess washing, the MAP resin was
resuspended in a DMF/PBS solution, then mixed with a 4-fold excess
of sulfosuccinimidyl (4-iodoacetyl amino benzoate (sulfo-SIAB)
activated oligosaccharides from S. pneumoniae serotype 14 (Os14).
After overnight mixing at room temperature, the MAP resin was
collected by filtration and washed with PBS, DMF and then methanol.
The MAP-Os14 resin was subjected to Ellman's test and sulfhydryl
group determination. The level of SH substitution was found to be
half of the starting value. Recoupling did not increase the amount
of Os14 conjugated to the MAP resin. The presence of
N-acetylgalactosamine (GlcNAc) in the glyco-MAP resin, a
carbohydrate found in Os14, was independently confirmed by
carbohydrate analysis. MAP-Os14 was first treated with 1% TFA to
remove Mtt (a lysine-protecting group) from Mtt-lysine residues,
then neutralized with a mild base, 1% diisopropylethylamine
(DIEA)/DMF. The presence of free amino groups was assayed by the
ninhydrine test which indicated that over 90% of Mtt groups had
been removed. The MAP-Os14 resin was resuspended in PBS, and then
mixed with four equivalents of periodate-oxidized S. pneumoniae
serotype 6B oligosaccharides (Os6B) in DMF/phosphate buffer (pH
7.8) in the presence of NaCNBH.sub.3 at 38.degree. C. for 6 days.
After conjugation, the substitution of amino groups was determined
by the ninhydrin assay was found to be 80 to 90% of the original
value. Again a double and triple coupling did not improve the
conjugation of Os6B to the MAP-Os14 resin. Although the coupling
efficiency was poor (about 15%), the presence of ribitol in the MAP
conjugate, a carbohydrate found in Os6B, was confirmed by
carbohydrate analysis.
[0086] The MAP-Os14-Os6B conjugate was treated with 20% piperidine
in DMF to remove the Fmoc protecting group from Fmoc-lysine
residues. After washing, the MAP-Os14-Os6B resin were mixed with a
4-fold excess of periodate oxidized S. pneumoniae serotype 19F
oligosaccharides (Os19F) in DMF/phosphate buffer (pH 7.8) in the
presence of NaCNBH.sub.3 at 38.degree. C. for 6 days. After
conjugation, the degree of amino group substitution measured by the
ninhydrin assay was found to be about 90%. The coupling reaction
was repeated and its efficiency was determined to be about 15%.
However, the presence of N-acetylmannose (ManNAc), a sugar found in
Os19F, was detected by carbohydrate analysis. A small portion of
MAP glycoconjugate-resin was cleaved with 95% TFA in the presence
of EDT and thioanisol. After the work-up, the MAP-glycoconjugate
was assayed for amino acid composition and carbohydrate content,
and found to have a correct amino acid composition and a correct
carbohydrate content. Although the overall yield was very low
(about 5%), these results nevertheless demonstrate that different
oligosaccharides can be selectively and sequentially conjugated to
a MAP resin. Furthermore, rabbit immunogenicity studies indicated
that this MAP glycopeptide conjugate was immunogenic and elicited
strong antibody responses against polysaccharides 19F and 14 (GMT
about 3000), but very weak anti-6B IgG responses (FIG. 11). The
antibody titers against 19F and 14 polysaccharides were significant
lower than those obtained in rabbits immunized with multivalent
oligosaccharides conjugated to TT (FIG. 7), but we still expect
that the pneumococcal multivalent MAP conjugate candidate vaccine
will be useful for human immunization. In addition, the rabbit
antisera reacted strongly with the T-cell peptides in
peptide-specific ELISAs (Table 3).
[0087] Utility of Synthetic Glycopeptide Conjugation
Technology.
[0088] In preferred embodiments of the present invention, the
glycoconjugate technology can be generally utilized to prepare
conjugate vaccines against pathogenic encapsulated bacteria. Thus,
the glycoconjugate technology of the present invention may be
applied to vaccinations to confer protection against infection with
any bacteria expressing potential protective polysaccharide
antigens, including Haemophilus influenzae, Streptococcus
pneumoniae, Escherichia coli, Neisseria meningitidis, Salmonella
typhi, Streptococcus mutans, Cryptococcus neoformans, Klebsiella,
Staphylococcus aureus and Pseudomonas aerogenosa.
[0089] In particular embodiments, the synthetic glycoconjugate
technology can be applied to produce vaccines eliciting antibodies
against proteins and oligosaccharides, such as Globo H, Le.sup.Y
and STn. Such vaccines may be used, for example, to induce immunity
against tumor cells, or to produce anti-tumor antibodies that can
be conjugated to chemotherapeutic or bioactive agents.
[0090] It is also understood that within the scope of the invention
are any variants or functionally equivalent variants of the above
specific peptides. The terms "variant" or "functionally equivalent
variant" as used above, mean that, if the peptide is modified by
addition, deletion or derivatization of one or more of the amino
acid residues, in any respect, and yet acts in a manner similar to
the specific peptides described herein, then such modified peptide
falls within the scope of the invention. Given the amino acid
sequence of these peptides (Table 1) and any similar peptide, these
are easily synthesized employing commercially available peptide
synthesizers, such as the Applied Biosystems Model 430A, or may be
produced by recombinant DNA technology.
[0091] It is clearly appears to one skilled in the art that the
various embodiments of the present invention have many applications
in the fields of vaccination, diagnosis and treatment of infection
and the generation of immunological reagents. A further
non-limiting discussion of such uses is further presented
below.
[0092] Vaccine preparation and use
[0093] As indicated above, the present invention, in one
embodiment, provides multivalent immunogenic conjugates useful for
formulating immunogenic compositions, suitable to be used as, for
example, vaccines. The immunogenic composition elicits an immune
response by the host to which it is administered including the
production of antibodies by the host.
[0094] The immunogenic compositions may be prepared as injectables,
as liquid solutions or emulsions. The antigens and immunogenic
compositions may be mixed with physiologically acceptable carriers
which are compatible therewith. These may include water, saline,
dextrose, glycerol, ethanol and combinations thereof. The vaccine
may further contain auxiliary substances, such as wetting or
emulsifying agents or pH buffering agents, to further enhance their
effectiveness. Vaccines may be administered by injection
subcutaneously or intramuscularly.
[0095] Alternatively, the immunogenic compositions formed according
to the present invention, may be formulated and delivered in a
manner to evoke an immune response at mucosal surfaces. Thus, the
immunogenic composition may be administered to mucosal surfaces by,
for example, the nasal or oral (intragastric) routes.
Alternatively, other modes of administration including
suppositories may be desirable. For suppositories, binders and
carriers may include, for example, polyalkylene glycols and
triglycerides. Oral formulations may include normally employed
incipients, such as pharmaceutical grades of saccharine, cellulose
and magnesium carbonate.
[0096] These compositions may take the form of solutions,
suspensions, tablets, pills, capsules, sustained release
formulations or powders and contain 1 to 95% of the immunogenic
compositions of the present invention.
[0097] The immunogenic compositions are administered in a manner
compatible with the dosage formulation, and in such amount as to be
therapeutically effective, protective and immunogenic. The quantity
to be administered depends on the subject to the immunized,
including, for example, the capacity of the subject's immune system
to synthesize antibodies, and if needed, to produce a cell-mediated
immune response. Precise amounts of antigen and immunogenic
composition to be administered depend on the judgement of the
practitioner. However, suitable dosage ranges are readily
determinable by those skilled in the art and may be of the order of
micrograms to milligrams. Suitable regimes for initial
administration and booster doses are also variable, but may include
an initial administration followed by subsequent administrations.
The dosage of the vaccine may also depend on the route of
administration and will vary according to the size of the host.
[0098] The concentration of antigen in an immunogenic composition
according to the invention is in general 1 to 95%. A vaccine which
contains antigenic material of only one pathogen is a monovalent
vaccine. Vaccines which contain antigenic material of several
pathogens are combined vaccines and also belong to the present
invention. Such combined vaccines contain, for example, material
from various pathogens or from various strains of the same
pathogen, or from combinations of various pathogens.
[0099] Immunogenicity can be significantly improved if the antigens
are co-administered with adjuvants, commonly used as 0.005 to 0.5
percent solution in phosphate buffered saline. Adjuvants enhance
the immunogenicity of an antigen but are not necessarily
immunogenic themselves. Adjuvants may act by retaining the antigen
locally near the site of administration to produce a depot effect
facilitating a slow, sustained release of antigen to cells of the
immune system. Adjuvants can also attract cells of the immune
system to an antigen depot and stimulate such cells to elicit
immune response.
[0100] Immunostimulatory agents or adjuvants have been used for
many years to improve the host immune responses to, for example,
vaccines. Intrinsic adjuvants, such as lipopolysaccharides,
normally are the components be the killed or attenuated bacteria
used as vaccines. Extrinsic adjuvants are immunomodulators which
are typically noncovalently linked to antigens and are formulated
to enhance the host immune responses. Thus, adjuvants have been
identified that enhance the immune response to antigens delivered
parenterally. Some of these adjuvants are toxic, however, and can
cause undesirable side effects, making them unsuitable for use in
humans and many animals. Indeed, only aluminum hydroxide and
aluminum phosphate (collectively commonly referred to as alum) are
routinely used as adjuvants in human and veterinary vaccines. The
efficacy of alum in increasing antibody responses to diphtheria and
tetanus toxoids is well established and, more recently, a HBsAg
vaccine has been adjuvanted with alum. While the usefulness of alum
is well established for some applications, it has limitations. For
example, alum is ineffective for influenza vaccination and
inconsistently elicits a cell mediated immune response. The
antibodies elicited by alum-adjuvanted antigens are mainly of the
IgGl isotype in the mouse, which may not be optimal for protection
by some vaccinal agents.
[0101] A wide range of extrinsic adjuvants can provoke potent
immune responses to antigens. These include saponins complexed to
membrane protein antigens (immune stimulating complexes), pluronic
polymers with mineral oil, killed mycobacteria in mineral oil,
Freund's complete adjuvant, bacterial products, such as muramyl
dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A,
and liposomes.
[0102] To efficiently induce humoral immune responses (HIR) and
cell-mediated immunity (CMI), immunogens are often emulsified in
adjuvants. Many adjuvants are toxic, inducing granulomas, acute and
chronic inflammations (Freund's complete adjuvant, FCA), cytolysis
(saponins and Pluronic polymers) and pyrogenicity, arthritis and
anterior uveitis (LPS and MDP). Although FCA is an excellent
adjuvant and widely used in research, it is not licensed for use in
human or veterinary vaccines because of its toxicity.
[0103] Desirable characteristics of ideal adjuvants include:
[0104] (1) lack of toxicity;
[0105] (2) ability to stimulate a long-lasting immune response;
[0106] (3) simplicity of manufacture and stability in long-term
storage;
[0107] (4) ability to elicit both CMI and HIR to antigens
administered by various routes;
[0108] (5) synergy with other adjuvants;
[0109] (6) capability of selectively interacting with populations
of antigen presenting cells (APC);
[0110] (7) ability to specifically elicit appropriate T.1 or TH2
cell-specific immune responses; and
[0111] (8) ability to selectively increase appropriate antibody
isotype levels (for example, IgA) against antigens.
[0112] U.S. Pat. No. 4,855,283 granted to Lockhoff et al on Aug. 8,
1989 which is incorporated herein by reference thereto teaches
glycolipid analogues including N-glycosylamides, N-glycosylureas
and N glycosylcarbamates, each of which is substituted in the sugar
residue by an amino acid, as immune-modulators or adjuvants. Thus,
Lockhoff et al. (U.S. Pat. No. 4,855,283 and ref. 29) reported that
N-glycolipid analogs displaying structural similarities to the
naturally occurring glycolipids, such as glycosphingolipids and
glycoglycerolipids, are capable of eliciting strong immune
responses in both herpes simplex virus vaccine and pseudorabies
virus vaccine. Some glycolipids have beensynthesized from long
chain alkylamines and fatty acids that are linked directly with the
sugars through the anomeric carbon atom, to mimic the functions of
the naturally occurring lipid residues.
[0113] U.S. Pat. No. 4,258,029 granted to Moloney, assigned to the
assignee hereof and incorporated herein by reference thereto,
teaches that octadecyl tyrosine hydrochloride (OTH) functions as an
adjuvant when complexed with tetanus toxoid and formalin
inactivated type I, II and III poliomyelitis virus vaccine. Also,
Nixon-George et al. (ref. 30), reported that octodecyl esters of
aromatic amino acids complexed with a recombinant hepatitis B
surface antigen, enhanced the host immune responses against
hepatitis B virus.
[0114] Immunoassays
[0115] In one embodiment, the conjugates of the present invention
are useful for the production of immunogenic compositions that can
be used to generate antigen-specific antibodies that are useful in
the specific identification of that antigen in an immunoassay
according to a diagnostic embodiment. Such immunoassays include
enzyme-linked immunosorbent assays (ELISA), RIAs and other
non-enzyme linked antibody binding assays or procedures known in
the art. In ELISA assays, the antigen-specific antibodies are
immobilized onto a selected surface; for example, the wells of a
polystyrene microtiter plate. After washing to remove incompletely
adsorbed antibodies, a nonspecific protein, such as a solution of
bovine serum albumin (BSA) or casein, that is known to be
antigenically neutral with regard to the test sample may be bound
to the selected surface. This allows for blocking of nonspecific
adsorption sites on the immobilizing surface and thus reduces the
background caused by nonspecific bindings of antigens onto the
surface. The immobilizing surface is then contacted with a sample,
such as clinical or biological materials, to be tested in a manner
conducive to immune complex (antigen/antibody) formation. This may
include diluting the sample with diluents, such as BSA, bovine
gamma globulin (BGG) and/or phosphate buffered saline (PBS)/Tween.
The sample is then allowed to incubate for from about 2 to 4 hours,
at temperatures such as of the order of about 25.degree. to
37.degree. C. Following incubation, the sample-contacted surface is
washed to remove nonimmunocomplexed material. The washing procedure
may include washing with a solution, such as PBS/Tween or a borate
buffer.
[0116] Following formation of specific immunocomplexes between the
antigen in the test sample and the bound antigen-specific
antibodies, and subsequent washing, the occurrence, and even
amount, of immunocomplex formation may be determined by subjecting
the immunocomplex to a second antibody having specificity for the
antigen. To provide detecting means, the second antibody may have
an associated activity, such as an enzymatic activity, that will
generate, for example, a colour development upon incubating with an
appropriate chromogenic substrate. Quantification may then achieved
by measuring the degree of colour generation using, for example, a
visible spectra spectrophotometer. In an additional embodiment, the
present invention includes a diagnostic kit comprising
antigen-specific antibodies generated by immunization of a host
with immunogenic compositions produced according to the present
invention.
[0117] It is understood that the application of the methology of
the present invention is within the capabilities of those having
ordinary skills in the art. Examples of the products of the present
invention and processes for their preparation and use appear in the
following examples.
EXAMPLES
[0118] The above disclosure generally describes the present
invention. A more complete understanding can be obtained by
reference to the following specific Examples. These Examples are
described solely for purposes of illustration and are not intended
to limit the scope of the invention. Changes in form and
substitution of equivalents are contemplated as circumstances may
suggest or render expedient. Although specific terms have been
employed herein, such terms are intended in a descriptive sense and
not for purposes of limitations. Immunological methods may not be
explicitly described in this disclosure but are well within the
scope of those skilled in the art.
Example 1
[0119] Preparation of acid-hydrolyzed group B meningococcal (GBM)
oligosaccharides.
[0120] This Example describes a method for preparing GBM
oligosccharides (M. wt. 3000 to 4500 Da) from the commercially
available GBM polysaccharides (M. wt>10 KDa).
[0121] Reagents required:
[0122] 1-GBM polysaccharides from Sigma cat # C-5762.
[0123] 2-Sodium acetate (50 mM) buffer pH 5.00, prepared by mixing
one volume of 0.5M sodium acetate with one volume of 0.23M acetic
acid.
[0124] 3-Reaction vial and a magnetic stirring bar.
[0125] 4-Sephadex G-25 gel column
[0126] 5-Ammonium bicarbonate (20 mM)
[0127] Procedure:
[0128] The GBM polysaccharide (200 mg) was dissolved in 15 mL of
degassed 50 mM sodium acetate buffer, pH 5.0 and the mixture was
then stirred at 80.degree. C. for 1 hr. The reaction mixture was
then immediately cooled with ice and neutralized to pH 7.0 by
dropwise addition of 0.1M NaOH. The total mixture was then
lyophilized to yield a crude product (460 mg, containing sodium
acetate) About 100 mg acid treated GBM were first dissolved in 3 mL
of 20 mM ammonium bicarbonate and then loaded into a Sephadex G-25
gel column equilibrated with 20 mM ammonium bicarbonate solution
using the following conditions:
[0129] Column: (10.times.1000 mm), calibrated with dextran 8800,
.beta.-cyclodextran and sucrose standards.
[0130] Flow rate: 0.6 mL/min.
[0131] Buffer: 20 mM ammonium bicarbonate.
[0132] Fraction collected at 4.5 min/tube.
[0133] The fractions were assayed for the presence of sialic acid
using the resorcinol/sulfuric acid assay (ref. 12). The elution
profile was then plotted and sialic acid-containing fractions with
an average molecular weight of 4 kDa were pooled and lyophilized.
The final yield of the acid hydrolyzed GBM was obtained.
Example 2
[0134] This Example shows chemical modification of acid-hydrolyzed
GBM oligosaccharides.
[0135] N-propionylated, acid-hydrolyzed GBM oligosaccharides were
prepared according to the method previously described by H.
Jennings et al. (ref. 6) with some modifications. The
N-propionylated GBM oligosaccharides ultimately were coupled to a
MAP backbone containing other oligosaccharide to produce
multivalent multiple carbohydrate vaccines, as described below.
[0136] Reagents required:
[0137] 1-Acid-hydrolyzed GBM oligosaccharides.
[0138] 2-Sodium hydroxide (2M solution).
[0139] 3-Propionic anhydride (Aldrich).
[0140] 4-Ammonium bicarbonate (10 mM)
[0141] 5-Aqueous oxalic acid (50%)
[0142] 6-Sodium borohydride (Sigma)
[0143] Procedure:
[0144] N-deacetylation of the acid-hydrolyzed GBM
oligosaccharides:
[0145] N-deacetylated acid-hydrolyzed group B meningococcal
polysaccharides was prepared according to the method described by
Jenning et al., with three modifications;
[0146] 1-The reaction was performed at ca 110.degree. to
120.degree. C.
[0147] 2-The dialysis was performed using molecular porous membrane
(1000 mol. wt. cut off).
[0148] 3-The neutralization of sodium hydroxide was accomplished
using 50% aqueous oxalic acid in the cold and last over 1 hr.
[0149] The polysaccharide (100 mg) was dissolved in 5 mL of
degassed 2M sodium hydroxide containing sodium borohydride (10 mg).
The resulting mixture was then heated for 6 to 8 hours at about
100.degree. to 120.degree. C. and the product was isolated by a
combination of pH neutralization in an ice bath using oxalic acid
50%, followed by dialysis (four changes of 10 mM ammonium
bicarbonate, 4.degree. C.) and lyophilization to provide a product
(65.2 mg). This de-acetylation resulted in 100% de-acetylation, as
determined by complete disappearance of the acetyl signal in the
.sup.1H NMR spectrum.
[0150] Preparation of N-propionyl GBM oligosaccharides
[0151] The N-deacetylated GBM oligosaccharide prepared from the
previous step (55 mg) was dissolved in saturated sodium bicarbonate
(12 mL) and three aliquots of propionic anhydride (0.250 mL) were
added over 30 minutes period. The total mixture was then stirred
overnight at room temperature. Ninhydrine test was performed and
found to be negative indicating complete conversion of free amino
groups to propionamido groups. The mixture was then dialyzed
against distilled water (3.times.4 L) and lyophilized to afford the
acid-hydrolyzed propionylated GBM oligosaccharide (43.2 mg).
Example 3
[0152] This Example shows the preparation of Oligosaccharides from
Streptococcus Pneumoniae
[0153] This Example describes the general methods using acid
hydrolysis of Streptococcus pneumoniae capsular polysaccharides
(CP) (M. wt.about.50 kDa) to produce oligosaccharides with a
molecular mass ranging from 2.5 to 5.0 kDa. The resulting
oligosaccharides can be subjected to a novel glycoconjugation
technology to prepare glycoconjugates containing
multiple-oligosaccharides covalently linked to a carrier protein or
a multiple antigen peptide system (MAP).
[0154] Reagents required:
[0155] 1-CP serotypes 6B, 14, 19F and 23F (ATTC)
[0156] 2-Acetic acid
[0157] 3-Trifloroacetic acid
[0158] 4-Gel chromatography column (Sephadex G-100, 10.times.1000
mm)
[0159] 5-Round bottom flask (250 mL)
[0160] 6-Magnetic stirring bar
[0161] 7-Oil bath
[0162] Procedure:
[0163] In a round bottom flask, the CPs (see Table below) was
dissolved in warm degassed water (62.5 mL) followed by the addition
of the required amount of degassed acid (see below). The total
mixture was degassed for an additional 10 minutes then heated using
an oil bath for the required time (see below). At the end of the
hydrolysis time, the total mixture was diluted 5-fold with water
and then lyophilized to produce the crude product.
1 CPs 6B 14 19F 23F Amount of CPs 250 mg 250 mg 250 mg 230 mg in
water in water in water in water (62.5 mL) (62.5 mL) (62.5 mL)
(62.5 mL) Buffer (mL) 0.02 M acetic 1 M TFA 0.02 M 0.5 M TFA acid
acetic acid (62.5 mL, pH (62.5 mL) (62.5 mL) (62.5 mL) 3.22) Time
30 h 7 h 48 h 3 h Temperature 100 70 50 70 (.degree. C.) Crude
product 200 mg 260 mg 200 mg 250 mg (mg) Pure product 160 mg 230 mg
180 mg 188 mg (mg) M. wt. of 2330 5200 2930 4640 product
[0164] A gel permeation column (10.times.1000 mm, Sephadex.RTM. -
G100) was calibrated with the following molecular weight standards:
Dextran standards (M. wt 8800, 39100, 73500, 503,000), glucose
(180), sucrose (342) and synthetic PRP hexamer (2340). The
purification of oligosaccharides was accomplished using
Sephadex.RTM. G-100 gel column and oligosaccharides were eluted
with Milli-Q water at flow rate of 0.9 mL/min. The fractions were
collected every 3 minutes and assayed for the presence of
carbohydrates using phenol/sulfuric acid. The fractions containing
oligosaccharides with molecular weight 2.5 to 5 kDa were pooled and
lyophilized.
Example 4
[0165] This Example describes the preparation of Oligosaccharides
of N. meningiditis
[0166] As for the acid hydrolysis of pneumococcal CPs, the process
as applied to N. meningitidis involves acid hydrolysis,
lyophilization and purification using gel-filtration
chromatography. The conditions for acid hydrolysis of CPs from N.
meningococcal groups C, W-135 and Y were also optimized. Typically,
CPs (10 mg/mL) are mixed with 20 to 80 mM sodium acetate, pH 4.5 to
5.5, in sealed vials under argon at 65.degree. to 100.degree. C.
for 8 to 12 hours. Since group B CPs can undergo intramolecular
esterification under acidic conditions, hydrolysis was effected
under conditions used for CPs group C hydrolysis, but the
incubation time was limited to 1 hr and the pH of the reaction was
immediately adjusted to pH 7 with 0.1M NaOH to reverse the
esterification process (for details, see Example 1) Group A CPs
contain labile phosphodiester bonds, thus they were hydrolyzed
under mild acidic condition (such as 10 to 20 mM acetic acid) and
incubated at 50.degree. to 100.degree. C. for 30 to 48 hours. At
the end of each hydrolysis, the reaction solutions were diluted
5-fold with water and then lyophilized. The crude oligosaccharides
were fractioned by Sephadex.RTM. G-100 gel filtration
chromatography (2.times.210 cm, see above). Typical chromatographic
results are illustrated in FIG. 4. The fractions were assayed for
the presence of sialic acid using the resorcinol/sulfuric acid
assay (ref. 12). The elution profile was plotted, and
chromatographically purified oligosaccharides of 2 to 5 kDa were
pooled. Sized oligosaccharides typically contained 6 to 15
repeating units. The yields were 40 to 80%.
Example 5
[0167] This Example describes the preparation of multi-valent
oligosaccharides conjugated randomly to a carrier protein.
[0168] To illustrate a potential use of the present invention, S.
pneumoniae serotypes 6B, 14, 19F and 23F oligosaccharides were
randomly and covalently linked to TT as shown in FIG. 1. To a TT
solution (8 mg/1.2 mL of PBS), a 4 molar excess of
periodate-oxidized 6B (0.5 mg/0.1 mL PBS), 14 (1.4 mg/0.2 mL), 19 F
(0.65 mg/0.12 mL) and 23 F (1 mg/0.2 mL) oilgosaccharides were
added. The pH of the mixture was adjusted to 7.4 with a few drops
of 0.1M NaOH, and the reaction was stirred for 4 days at 37.degree.
C. At day 5, a 10-fold excess (100 .mu.L) of NaCNBH.sub.3 (5 mg/mL
) was added to the mixtures and stirred for another 3 days at
37.degree. C. The reaction mixture was then dialysed against excess
PBS to remove unreacted oligosaccharides and NaCNBH.sub.3 for 3
days at 4.degree. C. The glycoconjugate was purified by the
gel-filtration chromatography on a Sephedex G100 column
(1.6.times.100 cm). The elution profile is illustrated in FIG. 5.
The glycoconjugate was collected. Protein and carbohydrates
analyses were performed and the molar ratio of carbohydrate to
protein was found to be 7.1:1. The multiple antigenic
glycoconjugate (MAG) was used as an immunogen formulated either
with complete Freund's adjuvant or alum. Rabbit and mouse
immunogenicity studies were performed. The results are described
below.
Example 6
[0169] This Example describes peptide synthesis.
[0170] Peptides (Table 1) were synthesized using an ABI 430A
peptide synthesizer and optimized t-Boc chemistry as described by
the manufacturer, then cleaved from the resin by hydrofluoric acid
(HF). The peptides were purified by reverse-phase high performance
liquid chromatography (RP-HPLC) on a Vydac C4 semi-preparative
column (1.times.30 cm) using a 15 to 55% acetonltrile gradient in
0.1% trifluoryl acetic acid (TFA) developed over 40 minutes at a
flow rate of 2 mL/min. All synthetic peptides used in biochemical
and immunological studies were >95% pure as judged by analytical
HPLC. Amino acid composition analyses performed on a Waters
Pico-Tag system were in good agreement with the theoretical
compositions. The synthetic MAP was manually prepared using Fmoc
solid-phase peptide synthesis chemistry according to a modified
method previously described by Tam (ref. 24). A Fmoc-Lys(t-Boc)-TGA
resin (500 mg, purchased from BACHEM) with a substitution level of
180 .mu.mol/g was normal by used to prepare MAP. As a general
coupling protocol, a 4-fold excess of Fmoc-protected amino acids
activated with an equal amount of HBTU and HOBT/DIEA for 1 hr, was
used. In order to facilitate the conjugation with oligosaccharides,
the substitution level of MAP was reduced to about 50 .mu.mol/g
when the first Fmoc-Gly residue was coupled. When the synthesis was
completed according to FIG. 2, a small portion of MAP-resin was
cleaved with 95% TFA in the presence of ethane dithiol (EDT) and
thioanisol, and amino acid analysis of the cleaved MAP was
performed to confirm the amino acid composition.
Example 7
[0171] This Example describes preparation of oligosaccharides with
cross-linking bifunctional groups.
[0172] Attached an amino linker to oligosaccharides. To a
periodate-oxidized oligosaccharide solution (3 mg/mL of PBS), a 20
molar excess of 1,4-diaminobutane (10.5 mg) was added. The pH of
the mixture was adjusted to 7.4, and then the reaction was stirred
for 4 day at 37.degree. C. At day 5, an excess (500 .mu.L) of
NaCNBH.sub.3 (20 mg/mL) was added to the mixture which was stirred
for 3 more days at 37.degree. C. The oligosaccharide derivatized
with a functional amino group, was purified by gel-filtration
chromatography on a Sephedex.RTM. G-50 column (1.6.times.100
cm).
[0173] Attached a thio-directed cross-linker (MBS) to
oligosaccharides.
[0174] m-Maleimidobenzoyl-N-hydroxysuccinimide (MBS, Pierce) (20
mg; 63.6 mmol) in tetrahydrofuran (1 mL) was added to a solution of
amino-derivatized oligosaccharides (4.3 mmol) in 0.1M phosphate
buffer (1 mL), pH 7.5. The reaction mixture was stirred for 30 min
at room temperature under argon, then extracted with ether
(4.times.5 mL) to remove excess MBS. The resulting aqueous layer
was applied to a Sephadex G-25 column (20.times.300 mm)
equilibrated with 20 mM ammonium bicarbonate buffer, pH 7.2 and
eluted with the same buffer. Elution was monitored by absorbance at
280 nm, and the eluted peak was pooled and lyophilized to produce
the desired MBS activated oilogsaccharides. The number of maleimide
groups incorporated into the oligomers was determined by adding
excess 2-mercaptoethanol to the activated oligosaccaharide-MBS and
back-titrating the excess using a modified Ellman's method (ref.
25).
Example 8
[0175] This Example describes the preparation of linear
glycopeptide conjugates.
[0176] A Fmoc-Gly-Lys(t-Boc)-TGA resin (500 mg) with a substitution
level of 50 .mu.mol/g was used to prepare linear peptides
containing a T-cell epitope derived from either S. pneumoniae or N.
meningiditis proteins as shown in Table 1. A standard Fmoc
chemistry coupling protocol was used (see Example 6). When the
synthesis was completed, a small portion of peptide-resin was
cleaved with 95% TFA in the presence of EDT and thioanisol to
determine the quality of the synthesis. The rest of the
peptide-resin was first deprotected at the N-terminal using
piperidine, and then washed with dichloromethane, methanol, water,
and PBS. The PN(123-140) peptide-resin was mixed with
periodate-oxidized S. pneumoniae serotype 14 oligosaccharides (1
eq.) in methanol/phosphate buffer (pH 7.8) in the presence of
NaCNBH.sub.3 at 38.degree. C. for 6 days. After conjugation, the
degree of amino groups substitution was determined by the
ninhydrine assay and the total sugar content was assayed using the
orcinol test. The linear glycopeptide-resin was cleaved with 95%
TFA in the presence of EDT and thioanisol. After the work-up, the
glycopeptide was assayed for amino acid composition and
carbohydrate content.
Example 9
[0177] This Example describes the preparation of multivalent MAP
glycopeptide conjugates.
[0178] A MAP resin containing three different T-cell epitopes
[PN(123-140), PN(263-281) and SP37, Table 1] derived from S.
pneumoniae membrane proteins was synthesized using a
Fmoc-Gly-Lys-TGA resin with a substitution level of 50 mmol/g as
shown in FIG. 2. The whole synthesis was carried out manually using
an optimized Fmoc chemistry coupling protocol described above
(Example 6). When the synthesis was completed, a small portion of
MAP-resin was cleaved with 95% TFA in the presence of EDT and
thioanisol. The cleaved MAP was found to have the correct amino
acid composition by amino acid analysis. The MAP resin was reduced
with DTT to remove the t-butylthio protecting groups from the
cysteine residues. After excess washing, the MAP resin was
resuspended in a DMF/PBS solution, then mixed with a 4-fold excess
of sulfo-SIAB activated oligosaccharides from S. pneumoniae
serotype 14 (Os14). After overnight mixing at room temperature, the
MAP resin was collected by filtration and washed with PBS, DMF and
then methanol. The MAP-Os14 resin was subjected to Ellman's test
and sulfhydryl group determination. The level of SH substitution
was found to be half of the starting value. Recoupling did not
increase the amount of Os14 conjugated to the MAP resin. The
presence of N-acetylgalactosamine (GlcNAc) in the glyco-MAP resin,
a carbohydrate found in Os14, was independently confirmed by
carbohydrate analysis. MAP-Os14 was first treated with 1% TFA to
remove Mtt (a lysine-protecting group) from Mtt-lysine residues,
then neutralized with a mild base, 1% diisopropylethylamine
(DIEA)/DMF. The presence of free amino groups was assayed by the
ninhydrine test which indicated that >90% of Mtt groups had been
removed. The MAP-Os14 resin were resuspended in PBS, and then mixed
with 4 equivalent of periodate-oxidized S. pneumoniae serotype 6B
oligosaccharides (Os6B) in DMF/ phosphate buffer (pH 7.8) in the
presence of NaCNBH.sub.3 at 38.degree. C. for 6 days. After
conjugation, the substitution of amino groups was determined by the
ninhydrin assay was found to be 80 to 90% of the original value.
Again a double and triple coupling did not improve the conjugation
of Os6B to the MAP-Os14 resin. Although the coupling efficiency was
poor (about 15%), the presence of ribitol in the MAP conjugate, a
carbohydrate found in Os6B, was confirmed by carbohydrate analysis.
The MAP-Os14-Os6B conjugate was treated with 20% piperidine in DMF
to remove the Fmoc protecting group from Fmoc-lysine residues.
After washing, the MAP-Os14-Os-6B resin were mixed with a 4-fold
excess of periodate oxidized S. pneumoniae serotype 19F
oligosaccharides (Os19F) in DMF/phosphate buffer (pH 7.8) in the
presence of NaCNBH.sub.3 at 38.degree. C. for 6 days. After
conjugation, the degree of amino group substitution measured by the
ninhydrin assay was found to be about 90%. The coupling reaction
was repeated and its efficiency was determined to be about 15%.
However, the presence of N-acetylmannose (ManNAc), a sugar found in
Os19F, was detected by carbohydrate analysis. A small portion of
MAP glycoconjugate-resin was cleaved with 95% TFA in the presence
of EDT and thioanisol. After the work-up, the MAP-glycoconjugate
was assayed for amino acid composition and carbohydrate content,
and found to have a correct amino acid composition and a correct
carbohydrate content. Although the overall yield was very low
(about 5%), these results nevertheless demonstrate that different
oligosaccharides can be selectively and sequentially conjugated to
a MAP resin.
EXAMPLE 10
[0179] This Example describes the preparation of native
polysaccharide-polylysine conjugate.
[0180] A 0.5 mL of periodate-oxidized polysaccharides (25 mg in 1
mL of 0.1M sodium phosphate buffer, pH 6.0), prepared from native
S. pneumoniae or N. meningiditis polysaccharides treated with
aqueous sodium periodate, was added to polylysine (5 mg) in 2 mL of
0.2M sodium phosphate buffer, pH 8.0, followed by the addition of
sodium cyanoborohydride (10 eqv. to polylysine). After incubation
at 37.degree. C. for 5 days, the reaction mixture was dialysed
against 0.1M phosphate buffer (4.times.1 L), pH 7.5, and the
resulting solution was applied onto an analytical Superose 12
column (15.times.300 mm, Pharmacia) equilibrated with 0.2M sodium
phosphate buffer, pH 7.2, and eluted with the same buffer.
Fractions were monitored for absorbance at 230 nm. The major peak
was pooled. The amount of protein was determined using the Bio Rad
protein assay. The presence of polysaccharides was confirmed by the
orcinol test.
Example 11
[0181] This Example describes mouse immunogenicity studies of
multivalent oligosaccharides-TT conjugates.
[0182] Five mice (BALB/c) were immunized intramuscularly (im) with
multivalent oligosaccharide-TT conjugates (20 .mu.g of
oligosaccharides) emulsified in Freund's complete adjuvant (FCA),
and followed by two booster doses (half amount of the same
immunogen in incomplete Freund's adjuvant) at 2 week intervals.
Antisera were collected, inactivated at 56.degree. C., and then
stored at -20.degree. C.
Example 12
[0183] This Example describes rabbit immunogenicity studies of
multivalent oligosaccharides-TT conjugates formulated in alum.
[0184] Rabbits were immunized intramuscularly with 0.5 mL of
multivalent oligosaccharides-TT conjugates (20 .mu.g
oligosaccharides equivalent) mixed with 3 mg AlPO.sub.4 per mL,
followed by two booster doses (half amount of the same immunogen)
at 2 week intervals. Antisera were collected every 2 weeks after
the first injection, heat-inactivated at 56.degree. C. for 30 min
and stored at -20.degree. C.
Example 13
[0185] This Example describes rabbit immunogenicity studies of
multivalent oligosaccharides-carriers conjugates formulated in
FCA.
[0186] Rabbits were immunized intramuscularly with 0.5 mL of
multivalent oligosaccharides-TT or oligosaccharides-MAP conjugates
(conjugates containing 12 .mu.g oligosaccharides equivalent mixed
with 1 mL of FCA), followed by two booster doses (half amount of
the same immunogen formulated with Fruend's incomplete adjuvant
(IFA)) at 2 week intervals. Antisera were collected every 2 weeks
after the first injection, heat-inactivated at 56.degree. C. for 30
min and stored at -20.degree. C.
Example 14
[0187] This Example describes peptide-specific ELISAs
[0188] Microtiter plate wells (Nunc-Immunoplate, Nunc, Denmark)
were coated with 500 ng of individual peptides in 50 .mu.L of
coating buffer (15 mM Na.sub.2CO.sub.3, 35 mM NaHCO.sub.3, pH 9.6)
for 16 hr at room temperature. The plates were then blocked with
0.1% (w/v) BSA in phosphate buffer saline (PBS) for 30 min at room
temperature. Serially diluted antisera were added to the wells and
incubated for 1 hr at room temperature. After removal of the
antisera, the plates were washed five times with PBS containing
0.1% (w/v) Tween-20 and 0.1% (w/v) BSA. F(ab')2 from goat
anti-rabbit IgG antibodies conjugated to horseradish peroxidase
(Jackson ImmunoResearch Labs Inc., Pa.) were diluted (1/8,000) with
washing buffer, and added onto the microtiter plates. After 1 hr
incubation at room temperature, the plates were washed five times
with the washing buffer. The plates were then developed using
tetramethylbenzidine (TMB) in H.sub.2O.sub.2 (ADI, Toronto) as
substrate. The reaction was stopped with 1N H.sub.2SO.sub.4 and the
optical density was measured at 450 nm using a Titretek Multiskan
II (Flow Labs., Virginia). Two irrelevant pertussis toxin peptides
NAD-S1 (19 residues) and S3(123-154) (32 residues) were included as
negative controls in the peptide-specific ELISAs. Assays were
performed in triplicates, and the reactive titre of an antiserum
was defined as the dilution consistently showing a two-fold
increase in absorbance value over that obtained with the pre-immune
serum.
Example 15
[0189] This Example describes anti-polysaccharide antibody
measurement.
[0190] Microtiter plate wells (Nunc-Immunoplate, Nunc, Denmark)
were coated with 200 ng of S. pneumoniae or N. meningiditis
polysaccahrides-polylysine conjugates in 200 .mu.L of coating
buffer (15 mM Na.sub.2CO.sub.3, 35 mM NaHCO.sub.3, pH 9.6) for 16
hr at room temperature. The plates were then blocked with 0.1%
(w/v) BSA in phosphate buffer saline (PBS) for 30 min at room
temperature. Serially diluted antisera raised against PRP-carrier
conjugates were added to the wells and incubated for 1 hr at room
temperature. After removal of the antisera, the plates were washed
five times with PBS containing 0.1% (w/v) Tween-20 and 0.1% (w/v)
BSA. F(ab').sub.2 from goat anti-rabbit IgG or anti-mouse IgG
antibodies conjugated to horseradish peroxidase (Jackson
ImmunoResearch Labs Inc., Pa.) were diluted (1/8,000) with washing
buffer, and added onto the microtiter plates. After 1 h-incubation
at room temperature, the plates were washed five times with the
washing buffer. The plates were then developed using the substrate
tetramethylbenzidine (TMB) in H.sub.2O.sub.2 (ADI, Toronto), the
reaction was stopped with 1N H.sub.2SO.sub.4 and the optical
density was measured at 450 nm using a Titretek Multiskan II (Flow
Labs., Virginia). Assays were performed in triplicates, and the
reactive titre of an antiserum was defined as the dilution
consistently showing a two-fold increase in O.D. value over that
obtained with the pre-immune serum.
Example 16
[0191] This Example describes a proliferation assay for synthetic
T-cell epitopes.
[0192] T-cell epitope mapping was performed by priming BALB/c mice
with 5 .mu.g of individual carrier proteins. Three weeks later, the
spleens were removed and the splenocytes cultured in RPMI 1640
(Flow Lab) supplemented with 10% heat-inactivated fetal calf serum
(Gibco), 2 mM L-glutamine (Flow Lab), 100 U/mL penicillin (Flow
Lab), 100 .mu.g/mL streptomycin (Flow Lab), 10 unit/mL rIL-2 and 50
.mu.M 2-mercaptoethanol (sigma) for 5-7 days. Proliferative
responses of the primed splenocytes to the panel of peptides were
determined in a standard in vitro assay (ref. 26). Briefly,
10.sup.6 splenocytes were co-cultured in a 96-well microtiter plate
with 5.times.10.sup.5 irradiated (1700 Rad) fresh syngeneic spleen
cells used as source of antigen presenting cells (APC) in the
presence of increasing molar concentrations (0.03 to 3 .mu.M of
peptide dissolved in the culture medium without IL-2). Cultures
were kept for 40 hr in a humidified 5% CO.sub.2/air incubator
maintained at 37.degree. C. During the final 16 hr of culture, 0.5
.mu.Ci of [.sup.3H]-Tdr (5 Ci/mmol, NEN) was added to each wells.
The cells were then harvested onto glass fibre filters, and the
incorporation of .sup.3H-thymidine into cellular DNA was measured
in a scintillation .beta.-counter (Beckman). Results are expressed
as the mean of triplicate determinations performed for each peptide
concentration. The standard deviation was always <15%.
Proliferative responses were considered as positive when
.sup.3H-thymidine incorporation was three-fold above that obtained
with either irrelevant peptides or the culture medium.
SUMMARY OF THE DISCLOSURE
[0193] In summary of this disclosure, the present invention
provides certain novel multivalent immunogenic oligosaccharides as
well as novel conjugation procedures in their preparation, their
use as vaccines and their use in the provision of antibodies for
diagnostic use. Modifications are possible within the scope of this
invention.
2TABLE 1 POTENTIAL T-CELL EPITOPES FROM MENINGOCOCCAL AND
PNEUMOCOCCAL PROTEINS PEP- SEQ ID TIDES SEQUENCE COMMENTS NO: NMTB-
PFTISDSDSLEGGFYGPKGEEL- Bactericidal 1 P2 AGKFLSNNDKVAAVFG Epitope
NM- KAKSRIRTKISDFGSFIGFKGSE- Human T-cell 2 C1-1 DLGEGLKA epitope
NM- VPAQNSKSAYKPAYYTKDTNNN- Human T-cell 3 C1-2 LTLVPAVVGK epitope
NM- AEQKSAVTEYYLNHGEWPGNN- Adhesion 4 Pi-1 TSAGVASSSTIKGKYVKEV
Epitopes PN GVRGAVNDLLAKWHQDYGQG Pneumolysin 5 (123- (123-40) 140)
PN- GFEALIKGVKVAPQTEWKQIG Pneumolysin 6 (263- (263-81) 281) SP37
GIIYAKNIAKQLIAKDPKNKDF- 37kDa Protein 7 YEKNG (1-30) PSP-
IKEIDESESEDYAKEGFRAPLQSK- Protective 8 AA IDAKKAKLSKLEELSDKIDELDA-
Epitope of EIAKLEDQIKAAEENNNVEDY- PspA EKEG (C) (193-261)
[0194]
3TABLE 2 Anti-peptide antibody responses in rabbits immunized with
combined linear glycopeptide conjugates [6B-PN(123-140) +
14-PN(263-281) + 23F-PN(123-140)] Anti-peptide antibody titer.sup.a
Peptides titre.sup.b Pre-Immune Geometric mean PN(123-140) <100
12,800 PN(263-281) <100 3,200 .sup.aTotal anti-peptide antibody
responses were determined by peptide-specific ELISAs.
.sup.bAntisera were obtained from rabbits immunized.
[0195]
4TABLE 3 Anti-peptide antibody responses in rabbits immunized with
a MAP glycopeptide conjugates. Anti-peptide antibody titer.sup.a
Peptides titre.sup.b Pre-Immune Geometric mean PN (123-140) <100
633,400 PN (263-281) <100 12,800 SP37 <100 51,200 .sup.aTotal
anti-peptide antibody responses were determined by peptide-specific
ELISAs. .sup.bAntisera were obtained from rabbits immunized three
times with the MAP glycopeptide conjugate.
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